Currently, RNA is commonly prepared using guanidinium-based lysis followed by extraction with phenol-chloroform and precipitation with ethanol (Chomczynski et al. (1987) Analytical Biochemistry 162(1):156-159). For extraction of RNA from bacterial cells, enzymes (e.g., lysozyme), strong detergents (e.g., sodium dodecyl sulfate (SDS)), and chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA) are also commonly used (Heptinstall The Nucleic Acid Protocols Handbook. R. Rapley, Ed. Humana Press: 2000; pp 47-52). Extraction mixtures typically have high ionic strengths (e.g., greater than 500 mM) and require careful purification or strong dilution, for example, by washing or buffer exchanges because the reagents used in RNA extraction, such as SDS, chaotropic agents, organic solvents, or alcohols are strong inhibitors of enzymes used in many standard experimental protocols in which RNA is used, such as PCR, restriction digestion, or ligation. In addition, current RNA extraction methods are notoriously time-consuming, tedious, and involve toxic reagents.
Although alkali lysis is widely used for isolation of plasmid DNA from bacterial cells (Birnboim et al. (1979) Nucleic acids research 7(6):1513-1523), it is not commonly used in isolating RNA because of the instability of RNA at high pH (Freitas et al. (2007) Molecular Biotechnology 36(2):151-158; Farrell (2005) RNA methodologies. Academic Press, Inc., San Diego, Calif.). Some groups have explored alkali-based RNA extraction from pelleted bacterial cells, though without providing adequate RNase control (Liao et al. (2007) J. Mol. Diagn. 9(2):158-168; Bercovici et al. (2011) Analytical Chemistry 83(11):4110-4117). If residual RNase activity is not eliminated, degradation of RNA will continue after lysis. Others have observed the lability of RNA in RNase-rich matrices, such as whole blood, where insufficient RNase inactivation results in severe degradation of RNA. For example, isolated RNA could no longer be amplified after incubation in blood plasma for as little as 15 seconds (Tsui, et al. (2002) Clinical Chemistry 48(10):1647-1653). Thus, adequate inactivation of RNases during cell lysis is clearly needed when preparing RNA from RNase-rich sources.
Schultz et al. (U.S. Pat. No. 5,981,235) proposed deactivating residual RNases using alkaline protease. In the method of Schultz et al., the RNA sample is suspended in a solution containing water, buffer and a chelating agent. The pH is adjusted to a value above 10 by adding a solution of sodium hydroxide. An anionic detergent and alkaline protease are added, and the solution is incubated for 5-30 minutes until nucleases are degraded. The pH of the solution is then lowered, and the RNA is isolated from the solution by precipitation with alcohol or using paramagnetic particles or a resin matrix containing silica particles in the presence of a chaotropic salt. The main disadvantages of this method is that the long incubation times required for preparing RNA from RNase-rich sources cause substantial degradation of the extracted RNA, and residual protease activity may interfere with further use of the extracted RNA.
Thus, there remains a need for a convenient, efficient method of preparing RNA that is free from degradation and contaminants.